Botulism is caused by exposure to toxins produced by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote currently exists. Seven different BoNT serotypes have been discovered to date (BoNT/A-G), many having numerous additional BoNT subtypes. To protect against all of these diverse BoNT bioterror threats, expensive conventional small molecule drug development would need to be separately performed for each of the seven different drug targets and perhaps others. This challenge, together with other extreme hurdles confronting BoNT small molecule drug development, particularly complicates efforts to develop small molecule drugs to treat botulism. New therapeutic paradigms are urgently needed to counter the enormous risks associated with these easy to obtain, easy to produce and extremely dangerous bioterror agents. We have developed and then extensively optimized two distinct 'designer E3-ligase' agents that each accelerate the 'molecular cure' of neurons intoxicated by one of the two most dangerous Botulinum neurotoxins, serotypes A or B (BoNT/A, BoNT/B). These two lead agents consist of the F-box domain of TrCP fused to a camelid 'nanobody' domain with binding specificity for one of the BoNT proteases. These polypeptide agents, with a size less than 30 kDa, bind to the BoNT protease and cause its rapid, intraneuronal destruction leading to rapid recovery of the neuron. Because of the modular nature of this antidote, it will be simple and straightforward to develop similar agents to treat all other BoNT serotypes and subtypes simply by substituting the nanobody domains with another having the appropriate specificity. In this proposal, we will develop a general delivery vehicle to deliver our two lead agents to the cytosol of intoxicated neurons within botulism patients and perform pre-clinical evaluation. As the vehicle, we propose to use an atoxic Clostridial toxin-based neuronal delivery vehicle (TNDV) due to its highly evolved capability to enter the body, survive in serum and deliver enzyme activities to the cytosol of targeted cells. We will develop three different and proven TNDV systems, each having unique and compelling features, and then select the best vehicle(s) for further development and animal testing. The three TNDV systems will be modified, atoxic forms of: 1) BoNT serotype C; 2) C. difficile toxin B and; 3) Clostridial C2 toxin. If successful, it is expected that similar agents could be developed to target the accelerated turnover of virtually any cytosolic neuronal protein for research or therapeutic applications. Public Health Relevance: Botulinum neurotoxin is an extremely dangerous, CDC Category A biodefense threat that is widely available, easily produced, exceedingly toxic and for which no antidote is available. Furthermore, at least seven different toxin types exist, each constituting of different drug targets and thus requiring distinct, challenging and expensive small molecule drug development programs. In contrast, the innovative new platform for biomolecule therapeutics that we propose to develop has the potential to quickly lead to commercially viable antidotes for all BoNT serotypes.